JPS6222599B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention describes the immobilization of a special category of intracellular enzymes that are sensitive to glutaraldehyde.
-zation). Enzymes belonging to this category (discussed below) are often thiol enzymes, which are enzymes that have an -SH group at or in close proximity to the active site of the enzyme molecule, thus making it possible for the thiol-reactive It will be inactivated by a reactive reagent such as glutaraldehyde. A typical example is urease. Various methods for producing immobilized enzymes are known as Methods of Enzymology.
Enzymology), Volume 44 (1977) (Academic
Press). Most of these methods involve particulate carriers which have the inherent disadvantage of high cost carriers and the inability to avoid high dilution of enzyme activity. It is also described how to immobilize glucose isomerase by cross-linking glucose isomerase-containing cells with glutaraldehyde and further processing. See, eg, US Pat. No. 3,980,521. Glucose isomerase is a very insensitive enzyme to glutaraldehyde, so the immobilization yield is high with this immobilization. Glutaraldehyde is a preferred crosslinking agent for various reasons. It is recognized by health authorities and is the best known crosslinking agent today from a manufacturing and economic standpoint. However, the present invention relates to the immobilization of intracellular enzymes that are sensitive to glutaraldehyde.
hitherto undesired high degrees of dilution (dilu-
It has been difficult to immobilize this type of enzyme without tion). It has not been possible to use the glutaraldehyde method for immobilization, since the conventional glutaraldehyde treatment of this category of enzymes always results in an almost complete loss of enzyme activity. The immobilization of enzymes that are sensitive to glutaraldehyde is therefore a completely different situation than the immobilization of enzymes that are not sensitive to glutaraldehyde. As used herein, the term "glutaraldehyde-sensitive enzyme" refers to
2223340, by treatment of cells containing the enzyme with glutaraldehyde to be acceptable for industrial use (e.g. for use in a continuous process or in batches in containers). means a glutaraldehyde-sensitive enzyme that loses more than 75% of its original activity when forming a physically strong granular product. The object of the present invention is to use glutaraldehyde as a cross-linking agent to produce immobilized enzymes in a very high yield, at a relatively low price and with a relatively small dilution of enzyme activity. It is an object of the present invention to provide a method for immobilizing glutaraldehyde-sensitive enzymes, making it possible to produce products consisting of immobilized enzymes that are well suited for industrial applications. According to one aspect of the present invention, there is provided a method for immobilizing an intracellular glutaraldehyde-sensitive enzyme, the method comprising: immobilizing a microbial cell (described below) containing an intracellular glutaraldehyde-sensitive enzyme in an aqueous medium at a significant content. A method is provided which consists of reacting with glutaraldehyde in the presence of a polyamine having one amino group and isolating the resulting immobilized microbial cells. As used herein, the term "microbial cells" refers to whole microbial cells, homogenates that can be produced from whole microbial cells, and anything in between, i.e., partially ruptured ( ruptured) used to mean microbial cells. As used herein, the term "significant content of primary amino groups" refers to the significant content of primary amino groups relative to the total content of primary, secondary and tertiary amino groups. used to mean Advantageously, the content of primary amino groups exceeds 5%, preferably 20%, of the total content of primary, secondary and tertiary amino groups. Typically, the ratio between primary, secondary and tertiary amino groups can be 1:2:1 when branched polyethyleneimine is used as polyamine. It is assumed that the primary amino group is reactive with glutaraldehyde, thereby forming a stable crosslink, which is why the primary amino group is important. Experiments performed with polyethyleneimine modified by reacting the primary (and secondary amino) groups with an epoxy compound showed that such modified polyethyleneimine does not work. It should be understood that since the reaction is carried out in an aqueous medium, the polyamine used according to the invention should be water-soluble. Surprisingly, the presence of polyamines results in very high recoveries of enzyme activity. Without the polyamine, the yield of enzyme activity is on the order of magnitude of about 0-15%, whereas with polyamine the yield can be on the order of magnitude of 30-70%. No inert particulate carrier is required and therefore the dilution of enzyme activity is small. When carrying out immobilization, the polyamine is added to the microbial cells before or at the same time as the glutaraldehyde. If glutaraldehyde is initially added to the microbial cells, the recovery of enzyme activity in the immobilized product will depend on PH, temperature, concentration and similar parameters unless the polyamine is added very quickly after glutaraldehyde addition. will decrease. The relative amounts of microbial cells, glutaraldehyde and polyamines are such that the product consisting of immobilized enzyme is well suited for industrial purposes, i.e. high enzyme activity, high activity yield (compared to the total enzyme activity before immobilization). ), should be selected to have high enzymatic stability and high mechanical stability. A preferred embodiment of the invention consists of reacting whole microbial cells. A further preferred embodiment of the invention provides a glutaraldehyde-susceptible strain produced by the bacterial strain NRRL11240 (or any strain essentially the same as the same, including mutants and variants thereof). Penicillin V-acylase (penicillin V-
acylase). A further preferred embodiment of the present invention is the species Bacillus pasteurius.
urease produced by a strain belonging to the species Kluyveromyces fragilis, and lactase produced by a strain belonging to the species Leuconostoc oenos. malolactic enzyme (malolactic)
It consists of immobilization of enzyme). A preferred embodiment of the invention consists in the use of the aqueous portion of the fermentation broth as the aqueous medium. This embodiment consists of direct treatment of the fermentation broth with glutaraldehyde and thus constitutes a very simple and economical immobilization method. However, the cells can be separated from the fermentation broth, for example by centrifugation, and then suspended in water or a suitable buffer of pH 5-10, the aqueous part of this suspension being subsequently treated with glutaraldehyde. is an aqueous medium. Similarly, the aqueous portion of the cell sludge, which is separated from the fermentation broth by e.g. centrifugation and has a toothpaste-like consistency, is used as an aqueous medium which is subsequently treated with glutaraldehyde. can do. Preference is given to using polyalkyleneimines as polyamines, preferably branched polyalkyleneimines, in which the alkylene moiety contains 2 to 4 carbon atoms. Preference is given to using branched polyethyleneimines or polyalkylene polyamines as polyamines. In a more preferred embodiment of the present invention, 500 to
Branched polyethyleneimine with a molecular weight of 100,000 daltons is used as polyamine.
0.01:1 to 10:1, preferably 0.1:1 to 1:1
It is preferred to use a ratio of primary amine equivalents/aldehyde equivalents in the range of . A preferred embodiment of the invention consists of a sequence in which the polyamine is added to the aqueous medium along with the microbial cells, followed by the addition of glutaraldehyde. Preferably, in the method of the invention 0.01:
A ratio of glutaraldehyde/microbial cells (W/W) of 1 to 100:1, preferably 0.1:1 to 10:1 can be used. In a further preferred embodiment of the invention, the isolated immobilized cells are shaped, preferably by granulation or extrusion. In another embodiment, the washed and isolated fixed cells are dried, milled and sieved to a particle size in the range of 100-1000μ, preferably 200-700μ. According to a second aspect of the invention there is provided an immobilized enzyme product produced by the method of the invention. A third aspect of the invention consists in the use of an immobilized product according to the second aspect of the invention, whereby the immobilized product can be applied batchwise or continuously, preferably in the latter case. can be used in columns. A preferred embodiment of the use of an immobilized product according to the invention is the immobilized penicillin V produced by strain NRRL11240 (or a strain essentially the same as that, including mutants and variants thereof).
-consisting of the use of acylase, whereby immobilized penicillin V-acylase is used continuously. See the comparative experiments below to demonstrate the surprising activity-preserving effect of polyamines in combination with glutaraldehyde-sensitive enzymes. a Bacillus pasteuri
pasteurii) ATCC11859 (6200IU/at PH7
g) Spray-dried urease-containing cells from 2
g was suspended in 98 ml of water with 10 μM β-mercaptoethanol. The pH of the cell suspension is 1.6ml
of 25% glutaraldehyde was added with stirring, it was 7.7. After stirring for 20 minutes at room temperature
The pH value dropped to 6.9. The fixed cells were filtered and washed twice with a 0.5 ml solution of 1mM β-mercaptoethanol in water. After vacuum drying (approx.
90 min at 50 °C), 0.7 g of the immobilized preparation at 125
Measured under optimal conditions for a particle size of ~300μ
Obtained with an activity of 64 IU/g. This equates to an activity yield of 0.3%. b The same cell suspension as described in a above, but adjusted to PH 7 instead of the treatment described in a above.
10% polyethyleneimine (molecular weight 60000, Dow
Chemicals PEI600) 4 ml and then
2.75 ml of 25% glutaraldehyde was added. Stirring was continued for 60 minutes at room temperature. 0.75 g of fixed cells obtained after filtering, washing and drying as in a above had an activity of 5600 IU/g (125-300 μ), ie the activity yield was 34%. Definitions of units of initial enzymatic activity of enzymes in the following examples are shown in the table below. table Enzymes Determination of initial enzyme activity Urease PH-stat titration with 1N HCl at PH7.0 and 30°C. 3% (0.5M) urea in 0.2M phosphate buffer is used as substrate, one unit being 1 μmol of hydrolyzed urea/min. Penicillin V Acylase Incubation at 40°C using 3% Penicillin V in 0.2M phosphate buffer PH 7.5. 6-APA produced after removing the cells was analyzed by Balasingham et al. (1972), Biochem.
Determined using p-aminobenzaldehyde according to Biophys. Acta 276, 250-56. 1 unit = 1 μmol of 6-APA produced/min. Unfixed cells are first opened by treatment with approximately 8% 1-butanol in 0.1M phosphate buffer with pH 7 for 20 minutes at room temperature. 4.75% in milk buffer PH6.5
(W/V) Kluyveromyces fragilis lactase by lactose
fragilis lactase) at 37°C (Das grosse Melkerei Lexikon,
Schultz 1965, Vol.2, M-Zp.740). After removing the cells, the glucose produced was collected using Boehringer Mannheim's blood sugar reagent (Boehringer Mannheim's
Blood Sugar reagent) determined by (GOD-
Perid method). 1 unit = 1 μmol of glucose produced/min. Malolactic Enzyme 33mM Maleate in 0.1M Phosphate Buffer PH5 - Using 1.67mM NAD ⁺ and 0.7mM _MnCl2
Incubation at 30°C. After removing the cells, the lactate produced by Gutmann, I. and
Wahlefeld.AW (1974) in Method of
Enzymatic Analysis, 2nd ed.Vol3, (HU
Bergmeyer, ed.), Academic Press, p.1463―
Determined by LDH + NAD ⁺ according to 67. 1 unit = 1 μmol lactate produced/min. The invention is further illustrated by the following examples. The main details of the examples can be seen from the table below.

Table: In all of the following examples, the PH value of the polyethyleneimine was adjusted to about 7 before addition. However, satisfactory immobilized enzymes can also be obtained with polyethyleneimine using other PH values, for example between about 4 and 9. In addition, the Bacterial strains used below
NRRL11240, Kluyveromyces fragilis
ATCC34439 strain (NRRL Y1109 strain), Bacillus
pasteurii ATCC 11859 strain and Leucorostoc
oenos DSM20252 strains are NRRL and ATCC, respectively.
and a publicly known strain freely available from DSM. Example 1 Fermentation broth 1 of strain NRRL 11240 containing approximately 1% dry cells and 1.5 penicillin V acylase units/ml was added with 10% polyethyleneimine (branched molecular weight 60000 from Dow Chemicals, PEI 600).
Add 30 ml with stirring followed by 5 minutes later 25 ml of 25% glutaraldehyde. Stirring was continued for an additional 60 minutes at room temperature, then the fixed cells were separated, washed with phosphate buffer (50mMPH7) and air dried. 14.2g of dried fixed cells
It was obtained with an activity of 71 units/g for a particle size of 180-420μ, ie the activity yield was 65%. Example 2 Fermentation broth 1.5 (0.68 penicillin V acylase units/ml) of strain NRRL11240 was supplemented with 10% polyethyleneimine (branched from Dow Chemicals).
45 ml of molecular weight 600, PEI6) were added with stirring and at 4°C 30 ml of 25% glutaraldehyde.
Stirring was continued at reduced speed for an additional 60 minutes, then the multi-cell particles formed were filtered, washed with phosphate buffer PH7, and air dried. The dried precipitate (21 g) had a penicillin V acylase activity of 14.5 units/g for a particle size of 200-700 microns, ie the activity yield was 30%. Example 3 Fermentation broth 380 (2.5 penicillin V acylase units/ml) of strain NRRL 11240 is cooled to approximately 15°C and 40 ml of 10% polyethyleneimine (Polymin P, BASF) is added with stirring, followed by 25% glutaric acid. Aldehyde (Union Carbide)
Added 9.4. Stirring was then continued at reduced speed for an additional 45 minutes, after which beta-mercaptoethanol was added to a final concentration of 25 mM and the fixed cells were separated on a filter press. Granulate the wet filter cake and heat the granules to 50-55℃
and dried in a fluidized bed. 7.1 Kg of dry preparation (unfractionated particles) with 55 units/g are obtained, which corresponds to an active yield of 41%. Example 4 Same strain NRRL11240 as described in Example 3
A series of immobilized preparations from fermentation broths of glutaraldehyde shows the effect of varying the concentration of polyethyleneimine on constant addition of glutaraldehyde. The results are shown in the table.

[Table] Polyamine P in the indicated quantities in each case
(BASF) was added as a 10% solution to 0.5 of the fermentation broth of strain NRRL 11240 (2.5 Penicillin V acylase units/g) with stirring, followed by the addition of 10 ml of 25% glutaraldehyde. Stirring was continued at reduced speed for an additional 60 min, after which cells were separated and 50mM
Dissolved in 25mM beta-mercaptoethanol in phosphate buffer PH7.5 and air dried. Approximately 9g
of dry material was obtained from each preparation. Example 5 To the fermentation broth of strain NRRL11240 (2.0 Penicillin V acylase units/ml) 200 ml of 10% polyethyleneimine (Polymin P, BASF) was added and the cells were then concentrated to approximately 1/10 of the original volume. 6 ml of 25% glutaraldehyde was added to the cell sludge with stirring and stirring continued at reduced speed for an additional 60 minutes. The fixed cells were then filtered and washed with 25mM beta-mercaptoethanol in 50mM phosphate PH7.5 on a glass strainer. Preparation 20 with 65 units/g (200-400μ) after air drying at room temperature
g was obtained. This equates to an activity yield of approximately 33%. A series of immobilized preparations made in a manner similar to that described above but using varying amounts of glutaraldehyde show the effect of varying the glutaraldehyde concentration after constant addition of polyethyleneimine and subsequent concentration. The results are shown in the table below.

TABLE Between 9 and 11 g of dry material was obtained from each preparation per fermentation broth. Example 6 350 fermentation broth of strain NRRL 11240 (2.4 penicillin V acylase units/ml) was cooled to about 15°C and 24.5 ml of 10% polyethyleneimine 24.5
[Taihei Sangyo Co., Ltd.
Kaisha) branched molecular weight approximately 700, PEI15T]
was added with stirring followed by 3.5 ml of 50% glutaraldehyde (Union Carbide). Stirring was then continued at reduced speed for an additional 45 minutes, after which beta-mercaptoethanol was added to a final concentration of 25 mM, and the fixed cells were separated by membrane press. Extrude the filter cake (500μ orifice) and add 50 particles
Dry in a fluidized bed at -55°C. The yield was 6.2 Kg of dry preparation with 85 Penicillin V acylase units/g (particle size <500 μ), which corresponds to an activity yield of 63%. Example 7 Same strain as in Examples 3 and 4
A fixed series of preparations from the fermentation broth of NRRL11240 shows the effect of using different types of polyethyleneimine.

【table】

[Table] In each case, add the indicated amount of polyethyleneimine to the bacterial strain while stirring as a 10% solution.
Fermentation broth of NRRL11240 0.5 (2.5 Penicillin V
acylase units/ml) followed by 10 ml of 25% glutaraldehyde. Stirring was continued at reduced speed for an additional 45 minutes. After that, separate the cells,
Washed with 25mM beta-mercaptoethanol in 50mM phosphate buffer PH7.5 and air dried. Example 8 10 g of fixed cells described in Example 1 were diluted with 50 mM
3% (w/v) crude K in phosphate buffer PH7.5
-Added to 200ml of penicillin V. Deacylation
Stirring speed of 150 rpm and constant pH of 7.5 (4N NaOH
(by titration) at 40°C.
95% of the penicillin was converted to 6-APA in 2.5 hours. This operation could be repeated with fresh Penicillin V at least 20 times without any substantial decrease in conversion. The degree of conversion was determined by HPLC (high pressure liquid chromatography), Waters' μBondapak, C-18.
Separation of the reaction mixture by column (elution with a gradient from 20% methyl alcohol in water to 50% methyl alcohol in water, ion pairing with Water's Pic A reagent and subsequent ultraviolet detection) determined by. Waters Associates′ information booklet
Paired―Ion Chromatography, D61 May1976,
Printed in USA (Maple Street, Milford,
See Mass.). 3% (W/V) K-penicillin V
The table shows the conversion results of 6-APA at 40°C.

Table: Example 9 9.7 g of fixed cells from Example 2 were added to 100 ml of 3% (w/v) K-penicillin V in 50 mM phosphate buffer PH 7.5. Stirring speed of 150rpm and
Deacylation was carried out at 40°C with a constant pH of 7.5 via PH-Stat. More than 90% of the penicillin was converted to 6-APA in 3 hours. After 10 sequential batches the conversion time increased to about 3.5 hours and after 20 batches to 4 hours. Example 10 Fixed cells corresponding to a dry weight of 100 g and originating from the product of Example 6 were dissolved in 50 mM at PH 7.5 containing 50 mM beta-mercaptoethanol.
It was reswollen in phosphate buffer and packed into a column with a diameter of 10 cm (bed volume 400 ml). Mercaptoethanol-containing buffer same as above
65 g of crude K-penicillin V in 1.5 was recycled through the preheater to the kalum (held at 35°C) and returned to the storage (held at 12°C) where it was
The PH was continuously adjusted to 7.5 by titration with 4M NaOH. At a circulation rate of 14 bed volumes per hour, 98% conversion of penicillin V to 6-APA was obtained after 4 hours. After 10 consecutive runs the reaction time had to be increased to 6 hours. Example 11 Kluyveromyces fragilis
- myces fragilis) ATCC34439 (NRRL
Y1109) fermentation broth (pH adjusted to 7.5) and 40 ml of 10% polyethyleneimine (branched molecular weight from Taihei Sangyo Kaisha) with stirring.
700, PEI15T) was added, followed by 16 ml of 25% glutaraldehyde. After 60 minutes, the particles were filtered, granulated, and air dried while stirring at room temperature. 6.5g of dry material obtained is approximately 500 units/g
lactase activity, which is approximately
Equivalent to 30%. Example 12 Bacillus pasteurii
ATCC11859 6x concentrated fermentation broth 300ml (6.3g
Dry cells, 9300 urease units/g) were added with 18 ml of 10% polyethyleneimine (Dow
Chemicals (branched molecular weight 60,000, PEI 600) was added followed by 20 ml of 25% glutaraldehyde. After stirring for an additional 60 minutes at room temperature, the fixed cells were separated and air-dried. dried fixed cells
13.8g is 1900 units/g for particle size of 300-700μ
was obtained with urease activity. This is about 45
% activity yield. Example 13 4% of Bacillus pasteurii ATCC 11859 with 230 urease units/ml
(W/V) 32 ml of 10% polyethyleneimine (Polymin HS, BASF) with stirring into 100 ml of aqueous suspension
was added, followed by 5 ml of 25% glutaraldehyde. Stirring was continued for 1 hour at 4°C, after which the cells were separated and air dried. 2.3g of dry preparation is 3100
Units/g of urease activity (particle size 300-700μ), which corresponds to an activity yield of 31%. Example 14 0.5 g of the immobilized cells described in Example 12 was packed into a column (1 cmφ x 10 cm), and the pH was adjusted to 9.
A substrate containing % urea (0.01% MgCl ₂ , 10 mM beta-mercaptoethanol) is passed through the bed at 40 °C, and 98% urea can be hydrolyzed at a flow rate of 50 ml/hr, with a half-life of enzymatic activity of approx. It was 1000 hours. Example 15 Fermentation broth of Leuconostoc oenos DSM20252 adjusted to pH 6.5 with NaOH with 14 units of malolactic activity/
To 0.25, 3 ml of 10% polyethyleneimine (Polymin P, BASF) was added with stirring, followed by 1
ml of 25% glutaraldehyde was added. Stirring was continued for an additional 30 minutes at 4°C, after which the fixed cells were separated, washed with 0.1M phosphate buffer PH6 and water, and then lyophilized. dried cells are
It has a malolactic activity of 3.4 units/g, which corresponds to an activity yield of about 40%. Example 16 1.6 moist cells of Leuconostoxenos DSM20252 with 96 units malolactic activity/g
g was suspended in 16 ml water and the pH was adjusted to 7.0.
1.7ml of 10% polyethyleneimine (Polymin P,
BASF) with stirring, followed by 1.5 ml of 25
% glutaraldehyde was added. Stir 1 at room temperature
This was continued for an hour, after which the fixed cells were filtered, washed with water, and air dried. Dry product (0.5g)
has a malolactic activity of 53 units/g, which corresponds to an activity yield of 28%. Example 17 4 g dry weight of the immobilized cells described in Example 15 were added to 50 ml of 25 mM L-maleate in 0.1 M phosphate at pH 5.0 along with 85 μmol NAD ⁺ and 35 μmol MnCl ₂ . Total conversion of maleate to lactate was achieved in 3 hours. This operation could be repeated at least 5 times without any increase in reaction time. Example 18 0.35 g of the preparation from Example 16 was added to 17 μmol NAD ⁺
and 7 μmol MnCl ₂ in 10 ml of 33 mM L-maleate in 0.1 M phosphate at PH 4.0. Total conversion of maleate to lactate was obtained in 6 hours and this operation could be repeated at least 5 times with fresh maleate, NAD ⁺ and MnCl ₂ without any increase in reaction time.

Claims (1)

[Scope of Claims] 1. A method for immobilizing an intracellular glutaraldehyde-sensitive enzyme, in which a microbial cell containing an intracellular glutaraldehyde-sensitive enzyme is immobilized with glutaraldehyde in the presence of a polyamine having a significant content of primary amino groups. A method characterized in that it consists of reacting with in an aqueous medium and isolating the immobilized enzyme obtained. 2. The method according to claim 1, wherein the microbial cells are whole microbial cells. 3. Claim 1 or 2, wherein the microbial cell is a microbial cell of strain NRRL11240 (or a strain essentially the same as that including mutants and variants thereof) and contains penicillin V-acylase. the method of. 4. The method according to claim 1 or 2, wherein the microbial cell is a microbial cell of a strain belonging to the species Bacillus pasteurii and contains urease. 5. The method according to claim 1 or 2, wherein the microbial cell is a microbial cell of a strain belonging to the species Kuriuberomyces fragilis and contains lactase. 6. The method according to claim 1 or 2, wherein the microbial cell is a microbial cell of a strain belonging to the species Leuconostocquenos and contains a malolactic enzyme. 7. A method according to any of claims 1 to 6, wherein the aqueous medium is the aqueous portion of the fermentation broth. 8. A method according to any one of claims 1 to 6, wherein the aqueous medium is the aqueous portion of a cell sludge, and the cell sludge is separated from the fermentation broth. 9. Claim 1, wherein the content of primary amino groups in the polyamine exceeds 5%, preferably exceeds 20% of the total content of primary, secondary and tertiary amino groups.
The method according to any one of items 1 to 8. 10 Claim 1, wherein the polyamine with a substantial content of primary amino groups is a polyalkylene imine whose alkylene portion contains 2 to 4 carbon atoms, preferably a branched polyalkylene imine. 9. The method according to item 9. 11. The method according to any one of claims 1 to 10, wherein the polyamine is a branched polyethyleneimine. 12. The method according to any one of claims 1 to 10, wherein the polyamine is a polyalkylene polyamine. 13 The branched polyethyleneimine is 500~
A method according to any of claims 1 to 11 having a molecular weight of 100,000 Daltons. 14 The ratio of primary amine equivalent/aldehyde equivalent is
0.01:1 to 10:1, preferably 0.1:1 to 1:1
14. A method according to any one of claims 1 to 13 within the scope of. 15. A method according to any of claims 1 to 14, wherein the method is carried out by a sequence consisting of adding the polyamine to an aqueous medium with microbial cells and then adding glutaraldehyde. 16 Glutaraldehyde/microbial cells (W/
16. A method according to any of claims 1 to 15, wherein the ratio of W) is in the range 0.01:1 to 100:1, preferably 0.1:1 to 10:1. 17. A method according to any of claims 1 to 16, wherein the isolated and immobilized enzyme is shaped, preferably by granulation or extrusion. 18 The washed and isolated immobilized enzyme is dried, milled and pulverized to 100-1000μ, preferably 200μ-
17. The method according to any one of claims 1 to 16, which is shaped by sieving to a particle size in the range of 700μ.